It is known that transitions between phases of the cell cycle are catalyzed by a family of cyclin-dependent kinases (Nurs, 1990; Hartwell et al., 1974). In many cells, transit through G1 of the cell cycle and entry into S phase requires a binding and activation of cyclin/cyclin-dependent kinase complexes (CDK), predominantly cyclin D-cdk4,6 and cyclin E-cdk2 (Sherr, 1994; Sherr, 1996).
The cyclin-dependent kinase inhibitors (CKIs) are naturally-occurring gene products which inhibit cyclin-CDK activity and phosphorylation of retinoblastoma protein (Rb), resulting in G1/S growth arrest (D. O. Morgan, 1995; Sherr and Roberts, 1995). CKIs directly implicated in CDK regulation are p21cip1/Waf1(Xiong et al., 1993; Harper et al., 1993), p27Kip1 (Pyoshima and Hunter, 1994; Polyak et al., 1994; Coats et al., 1996), and p16/p15INK4 (Serrano et al., 1993).
The ability of CKIs to arrest cells in G1 have made the proteins of particular use in gene therapy techniques for treating diseases or disorders associated with cell proliferation, such as cancer and leukemias, psoriasis, bone diseases, fibroproliferative disorders, atherosclerosis, restenosis, and chronic inflammation. However, very little is known about the regulation of these very important proteins in vivo.
The inventors have discovered a novel mechanism of regulation of CKIs. Specifically, disclosed herein are serine/threonine kinases that inhibit the ability of CKIs to arrest cells in G1. In light of this discovery, the inventors were able to construct a transdominant mutant of a serine/threonine kinase that interferes with the respective endogenous serine/threonine kinase when introduced into a cell transgenically. Furthermore, the inventors were able to construct a CKI unable to be inhibited by a serine/threonine kinase. Such constructs may be used alone, together, or in conjunction with other therapies for inhibiting or reducing cell proliferation.
Thus, the present invention provides isolated nucleic acid segments. Such isolated nucleic acid segments may encode wild-type or mutant hKIS polypeptides. In preferred embodiments, the isolated nucleic acid segments encode a transdominant mutant hKIS. A transdominant mutant hKIS is a polypeptide that is capable of interfering with the ability of endogenous hKIS to phosphorylate p27. Thus, a transdominant mutant hKIS would lead to or enhance cell cycle arrest in a cell containing the mutant. An example of a transdominant mutant hKIS is an hKIS that contains a mutation altering its serine/threonine kinase activity, such as that encoded by SEQ ID NO:3).
In other embodiments of the present invention, the isolated nucleic acid encodes a cyclin kinase inhibitor containing a mutation at a serine or threonine amino acid. It is preferred that the cyclin kinase inhibitor retains its ability to arrest the cell cycle. Examples of mutated cyclin dependent kinases include mutated of p16, p21, p27, and p57.
The isolated nucleic acids of the present invention may be contained in an expression vector. The expression vector may be a plasmid or a viral vector. The viral vector may be replication deficient and includes a retroviral vector, an adenoviral vector, an adenovirus associated viral vector, or a lentiviral vector.
Furthermore, the isolated nucleic acids of the present invention may be contained in or associated with a medical device, such as a catheter.
The isolated nucleic acids or polypeptides of the present invention may be included in a kit, such kits may also include one or more medical devices for administering the nucleic acid or polypeptide to a patient or one or more cells of a patient.